Method for determining glucose with o-toluidine reagent containing an arsenic compound

ABSTRACT

A DIAGNOSTIC C~MPOSITION CONTAINING O-TOLUIDINE, THIOUREA AND AN OXIDE OF ARSENIC IS USED FOR THE QUANTITATIVE DETERMINATION OF GLUCOSE IN THE BODY FLUIDS OF HUMANS.

United States Patent US. Cl. 23-230 B 4 Claims ABSTRACT OF THEDISCLOSURE A diagnostic composition containing o-toluidine, thiourea andan oxide of arsenic is used for the quantitative determination ofglucose in the body fluids of humans.

BACKGROUND OF THE INVENTION This invention relates to a new and improveddiagnostic composition which is useful for the quantitativedetermination of glucose in fluids, particularly body fluids such asurine, plasma, blood and the like.

The detection and quantitative determination of glucose in blood andurine is of great importance for diabetic patients whose diets must becontrolled so as to regulate sugar intake and who must frequently beguided in this regard by .regular checks on blood and urine glucose.Diabetes screening programs are dependent on the availability of rapid,inexpensive and accurate methods of glucose determination.

Procedures for the detection. of sugar in blood and urine are well knownin clinical chemistry. The Folin-Wu and Nelson-Somogyi proceduresutilize copper reduction for the detection and determination of sugar.US. Pat. 3,008,879 describes a means for testing biological fluids fortheir glucose content which involves a combination comprising an enzymesystem having glucose oxidase activity, a primary indicator materialwhich is capable of being preferentially oxidized to a colored form inthe presence of hydrogen peroxide and the material having peroxidativeactivity and, as a secondary indicator, a material which is capable ofreducing the preferentially oxidized primary indicator material whilesimultaneously becoming colored.

The use of o-toluidine solutions in glacial acetic acid for thedetermination of aldosaccharides in serum or plasma is described byHultman, E., Nature, 183, 108 (1959) and Dubowski, K. M. Clin. Chem. 8,215 (1962). Ordinarily, glucose is the only aldohexose present incli-nically significant amounts in 'blood serum; other aldohexoses thatmay be present in minute quantities are mannose and galactose. This testmethod is based on the formation of a colored complex of glucose ando-toluidine in glacial acetic acid. The concentration of this complex,and thus the intensity of the green color developed, is directlyproportional to the concentration of glucose present in the reactionmixture, and can be measured photometrically.

For the determination of glucose in blood, the Folin- Wu andNelson-Somogyi test methods can be applied only to protein-freefiltrates of blood serum or plasma. There is some loss of specificityand reliability because of saccharoids and other reducing compounds thatcannot be completely removed in preparation of a protein-free filtratewith resulting values higher than true glucose levels. Otherdisadvantages include the limited stability of the test reagents, theinstability of the final test color, and critical factors in the testprocedure involving temperature, heating time and alkalinity of reactionmixture.

The glucose-oxidase test is a highly specific test for glucose whencorrectly done with pure materials. A protein-free filtrate is required.Other disadvantages include the sensitivity and limited stability of thetest reagents, he necessary purity and standardization of theglucoseoxidase enzyme, the errors arising from interfering enzymeinhibitors, and the extreme care necessary during the three minuteheating process where a matter of seconds can critically aifect theaccuracy of results.

The o-toluidine test is a simple, rapid, reliable and economical methodfor glucose determination. Importantly, there is no need forprotein-free filtrates although the test can be performed with suchfiltrates. The test can be performed directly with blood serum orplasma. Orthotoluidine is the only reagent required. Acetic acidsolutions are stable for at least six months. This stability is enhancedby the addition of thiourea. The final test color has good stability. Animportant disadvantage, however, of o-toluidine is its susceptibility tooxidation with its resultant effect on test sensitivity. The depressedsensitivity of the reagent results in a regression line with a lowerslope when the optical densities of the final test colors are plottedagainst sugar concentrations, with serious loss of assay accuracy.

The object of this invention is to provide an improved diagnosticcomposition containing o-toluidine, thiourea and arsenic oxides for thesimple, rapid, inexpensive and accurate quantitative determination ofglucose in the biological fluids of humans.

SUMMARY OF THE INVENTION This invention is concerned with a diagnosticcomposition containing o-toluidine for the quantitative determination ofglucose in such biological fluids as blood and urine. It has been foundthat the incorporation of an oxide of arsenic in the o-toluidine reagentsolution increases the sensitivity of the test reagent and increases thestability of the final test color in the assay procedure.

DETAILED DESCRIPTION OF THE INVENTION The o-toluidine test method forthe quantitative determination of glucose in blood and urine involvesthe formation of a colored complex of glucose and o-toluidine in glacialacetic acid which follows Beers law. The green color, read at 630 nm.,is stable 97%) after standing one hour in air at 25 C.

o-Toluidine is relatively sensitive to oxidation. Bulk supplies ofuniform quality and purity are difficult to obtain, with EastmanChemical Company the preferred source. Freshly distilled o-toluidine is,of course, eminently suitable. The stability of o-toluidine solutions inglacial acetic acid (6.25% w./v.) is increased by the addition ofthiourea (1.50% w./v.). The addition of an arsenate salt or oxide ofarsenic increases the sensitivity of the o-toluidine reagent asreflected in the slope of the optical density curve of the final testcolors.

The arsenic compound may be selected from the group consisting of thefollowing:

sodium orthoarsenate: Na AsO 121-1 0 sodium mono-H-orthoa-rsenate: NaHAsO -7H O sodium mono-H-orthoarsenate: Na HAsO l2H O sodiumdi-H-orthoarsenate: NaH AsO -H O sodium metaarsenate: NaAsO sodiumarsenite: NaAsO- arsenic trioxide: AS203 arsenolite: AS405 claudetite:As O arsenic pentoxide: AS205 sodium thioarsenate: Na AsS -8H OTheaddition of sodium arsenate (Na HAsO -7H O) to the o-toluidinereagent increases the sensitivity to glucose up to An increase insensitivity, over solutions containing no additive, is observed evenwith small 0.1% w./v.) concentrations of sodium arsenate additive. The

sensitivity increase levels off in the region of 0.5% w./v. additive,and finally attains a value of 120% increase at saturated levels ofsodium arsenate (35% w./v.).

The color formed by the o-toluidine, with and without arsenate additive,is quite stable for the first hour. However, an increase in stability isnoted for the sodium arsenate additive solution after 15 hours with a32% decrease in color retained, compared to a 38% decrease for thecontrol solution. This increased color stability is important when largenumbers of test samples are run, and assay readings cannot beimmediately made.

The following examples are provided to illustrate the present invention,but not to limit its scope.

EXAMPLE I Preparation of reagent Thiourea (0.3 g., 1.5% w./v.) is placedin a 500 ml. Erlenmeyer flask and dissolved in 150 ml. of fresh glacialacetic acid. Fresh opened o-toluidine from Eastman Chemical Company(12.5 ml., 6.25% w./v.) is added dropwise to the stirred acetic acidsolution. Sodium mono- H-orthoarsenate, Na HAsO -7H O, (100 mg., 0.5%w./v.) is added all at once and dissolved by gentle stirring undernitrogen. The solution is stored at room temperaure in a dark glassbottle under nitrogen.

Test procedure (1) Using a suitable pipette add 0.05 ml. of blood serumor other sample to be tested to a 150 mm. x 15 mm., or similar, Pyrextest tube. If serum or plasma is used, deproteinization is notnecessary.

(2) Add 4.0 ml. of o-toluidine reagent to the tube. Mix sample andreagent thoroughly, preferably with a vortex mixer.

(3) In a similar manner, prepare a reagent blank using 0.05 ml. ofdistilled water instead of sample, and prepare a glucose standard using0.05 ml. of standard glucose solution instead of serum or other sample.

(4) Place the test tubes in a boiling water bath for exactly 10 minutes.

(5) Cool the solutions to room temperature, and determine opticaldensities at 630 nm.

(6) Determine the concentration of glucose in the sample from apreviously prepared curve of optical density vs. glucose concentration,using a series of at least three dilferent glucose concentrations in therange of expected results (50-400 mcgs. percent).

EXAMPLE II The preparation of the o-toluidine test reagent of Example Iis repeated in turn with sodium orthoarsenate (Na AsO -12H O), sodiummono-H-orthoarsenate (Na HAsO sodium di-R-orthoarsenate (NaH AsO -H O),sodium metaarsenate (NaAsO sodium arsenite (NaAsO arsenic trioxide (As oarsenolite (As O claudetite (ASaO arsenic pentoxide (AS305) and sodiumthio- 4 arsenate (Na AsS -8H 0) in place of sodium mono-H- orthoarsenate(Na HAsO -7H O), with comparable test results.

What is claimed is:

1. In the method for quantitatively determining glucose in thebiological fluids of humans using a diagnostic composition containingo-toluidine, the improvement which comprises incorporating an arseniccompound selected from the group consisting of sodium orthoarsenate (NaASO 12H O) sodium mono-H-orthoarsenate (Na HAsO -7H O), sodiummono-H-orthoarsenate (NaHAsO -12H O), sodium di-H-orthoarsenate (NaH AsO-H O), sodium metaarsenate (NaAsO sodium arsenite (NaAsO arsenictrioxide (As O arsenolite (AS406), claudetite (As O arsenic pentoxide(As O and sodium thioarsenate (Na AsS 2. The method of claim 1 whereinsaid arsenic compound is sodium mono-H-orthoarsenate (Na- HAsO (Na AsS8H O 4. The diagnostic composition of claim 3 wherein said arseniccompound is sodium mono-H-orthoarsenate (Na HAsO References Cited UNITEDSTATES PATENTS 3,001,915 9/1961 Fonner 23-253 TP 3,453,180 7/1969 Fraser23-253 TP 3,607,077 9/1971 Hartel 23-230 B 3,615,228 10/1971 Thiegs23230 B 3,653,836 4/ 1972 Gruher 23230 B 3,653,841 4/1972 Klein 23230 B3,660,559 5/1972 Buckert 23230 B MORRIS O. WOLK, Primary Examiner S.MARANTZ, Assistant Examiner US. Cl. X.R. 252-408

